Biocontrol of Storage Maladies of Potatoes by Bacterial Antagonists Produced in Co-Culture

ABSTRACT

Bacterial compositions effective for inhibiting fungal diseases of potatoes and/or potato sprouting are produced by co-culture of two or more of  Pseudomonas fluorescens  (NRRL B-21133),  Pseudomonas fluorescens  biovar (NRRL B-21053),  Pseudomonas fluorescens  (NRRL B-21102) and  Enterobacter cloacae  (NRRL B-21050). Compositions produced by co-culture of these bacteria together in the same culture medium are significantly more effective for inhibiting fungi-induced diseases of potatoes and/or inhibiting sprouting of potatoes, than blends or mixtures of the same bacteria cultured separately.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the biological control of fungal potatodiseases and/or potato sprout inhibition. More particularly, thisinvention relates to compositions of bacteria which are effectiveantagonists against fungal species responsible for Fusarium dry rot andother potato diseases which occur in the field or in postharveststorace.

2. Description of the Prior Art

The potato is the most important dicotyledonous source of human food,ranking as the fifth major food crop of the world. Fusarium-inducedpotato dry rot is an economically important problem of potatoes both inthe field and in storage. Several species of the Fusaria induce thisdisease, however, Gibberella pulicaris (Fries) Sacc. (anamorph: Fusariumsambucinum Fuckel) is a major cause worldwide, especially in NorthAmerica. Fusarium spp. can survive for years in field soil, but theprimary inoculum is generally borne on seed tuber surfaces. The dry rotfungi infect potatoes via wounds in the periderm inflicted duringharvesting or subsequent handling. In stored potatoes, dry rot developsmost rapidly in high relative humidity (circa 70% and higher) and at15°-20° C., but continues to advance at the coldest temperatures safefor potatoes. Although rots caused by Fusarium seldom reach epidemicproportions, the level of infected tubers in storage often reaches 60%or higher, with average losses estimated in the 10-20% range. Inaddition to destroying tissue, F. sambucinum can produce trichothecenesthat have been implicated in mycotoxicoses of humans and animals.

The high value of the potato crop and the significant economic lossescaused by potato dry rot have led to investigations of various methodsto control the disease. Success has been attained by use of thefungicides, thiabendazole and 2-aminobutane, which are applied to tubersat harvest or at preplanting [Carnegie et al. 1990. Ann App. Biol. 116:61-72; Leach, “Control of Postharvest Fusarium Tuber Dry Rot of WhitePotatoes,” pages 1-7 In ARS-NE-55, U.S. Dep. Agric., Washington, D.C.].However, strong concerns are being raised about the potential adverseimpact of these chemicals on ground and surface water reservoirs and onthe health of agricultural product workers and consumers. Also,thiabendazole-resistant strains of F. sambucinum have emerged inpopulations from severely dry-rotted tubers in North America and inEurope. Potato breeding programs have given increased attention todevelopment of cultivars with resistance to Fusarium but most of thereported cultivars produced by these programs are resistant to only oneor two of several Fusarium strains [Leach et al. 1981. Phytopathology71(6):623-629]

One alternative to chemical fungicides in controlling potato rot is theuse of biological agents. Postharvest biological control systems offruit have been actively investigated since the 1980's. These includeiturins as antifungal peptides in biological control of peach brown rotwith Bacillus subtilis [Gueldner et al. 1988. Journal of Agriculturaland Food Chemistry 36:366-370]; postharvest control of blue mold onapples using Pseudomonas spp. isolate L-22-64 or white yeast isolateF-43-31 [Janisiewicz, 1987. Phytopathology 77:481-485]; control of graymold of apple by Cryptococcus laurentii [Roberts, 1990. Phytopathology80:526-530]; biocontrol of blue mold and gray mold on apples using anantagonistic mixture of Pseudomonas spp. and Acremonium breve[Janisiewicz, 1988. Phytopathology 78:194-198]; control of gray mold andreduction in blue mold on apples and pears with an isolate ofPseudomonas capacia and pyrrolnitrin produced therefrom [Janisiewicz andRoitman, 1988. Phytopathology 78:1697-1700]; postharvest control ofbrown rot in peaches and other stone fruit with the B-3 strain ofBacillus subtilis [Pusey and Wilson. 1984, Plant Disease 68:753-756;Pusey et al. 1988. Plant Disease 72:622-626; and U.S. Pat. No. 4,764,371to Pusey et al.]; antagonistic action of Trichoderma pseudokoningiiagainst Botrytis cinerea Pers. which causes the dry eye rot disease ofapple [Tronsmo and Raa. 1977. Phytopathol. Z. 89:216-220; andpostharvest control of brown rot and Alternaria rot in cherries byisolates of Bacillus subtilis and Enterobacter aerogenes [Utkhede andSholberg. 1986. Canadian Journal of Microbiology 963-967]. A review ofbiological control of postharvest diseases of fruits and vegetables isgiven by Wisniewski et al. [1992. HortScience 27:94-98].

More recently, significant progress has also been made in the isolationand development of bacteria agents for controlling diseases of potatoes.For instance, eighteen Gram-negative bacteria were originally discoveredand developed as biocontrol agents to protect potatoes entering storagefrom Fusarium dry rot incited by Gibberella pulicaris [Schisler andSlininger. 1994. Selection and performance of bacterial strains forbiologically controlling Fusarium dry rot of potatoes incited byGibberella pulicaris. Plant Disease 8:251-255; Slininger et al. 1994Two-dimensional liquid culture focusing: A method of selectingcommercially promising microbial isolates with demonstrated biologicalcontrol capability, in: M H Ryder, P M Stephens and G D Bowen (Eds.).Improving plant productivity with rhizosphere bacteria, 3rdinternational workshop on plant growth-promoting rhizobacteria,Adelaide, S. Australia, pp. 29-32. Glen Osmond, South Australia CSIRODivision of Soils; Slininger et al. 1996. Bacteria for the control ofFusarium dry rot of potatoes. U.S. Pat. No. 5,552,315; Schisler et al.1997. Effects of antagonist cell concentration and two-strain mixtureson biological control of Fusarium dry rot of potatoes. Phytopathology87:177-183; Schisler et al. 1998. Bacterial control of Fusarium dry rotof potatoes. U.S. Pat. No. 5,783,411], and these bacteria significantlyreduced the level of dry rot disease in pilot trials (Schisler et al.2000. Biological control of Fusarium dry rot of potato tubers undercommercial storage conditions. American Journal of Potato Research77:29-40). Top dry rot suppressive strains included Pseudomonasfluorescens biovar 5 (S11:P:12 NRRL B-21133 and P22:Y:05 NRRL B-21053,Pseudomonas fluorescens biovar 1 (S22:T:04 NRRL B-21102) andEnterobacter cloacae (S11:T:07 NRRL B-21050). All of these strains werealso documented to suppress sprouting (Slininger et al. 2000. Biologicalcontrol of sprouting in potatoes. U.S. Pat. No. 6,107,247; Slininger etal. 2003. Postharvest biological control of potato sprouting by Fusariumdry rot suppressive bacteria. Biocontrol Science and Technology13:477-494), with Pseudomonas fluorescens S11:P:12 (NRRL B-21133)exhibiting the greatest efficacy, and E. cloacae S11:T:07 being thesecond most efficacious. In addition, the strains have been shown tosuppress late blight incited by Phytophthora infestans US-8 mating typeA2 in laboratory bioassays and small pilot simulations of commercialstorage conditions with top performance shown by the followingtreatments: a mixture of four strains (comprised ofS11:P:12+P22:Y:05+S22:T:04+ S11:T:07)>strain S22:T:04 used alone >strainS11:P:12 used alone [Slininger et al. 2007. Biological control ofpost-harvest late blight of potatoes. Biocontrol Science and Technology17(5/6):647-663]. Most recently, we showed the ability of several of thedry rot antagonistic bacteria to suppress pink rot disease incited byPhytophthora erythroseptica, including S11:T:07 which exhibited thegreatest efficacy and S22:T:04 exhibiting the third greatest efficacy(Schisler et al. 2007. Gram negative bacteria for reducing pink rot, dryrot, late blight, and sprouting potato tubers in storage. AmericanJournal of Potato Research 84:115; Schisler et al. 2009. Bacterialantagonists, zoospore inoculums retention time and potato cultivarinfluence pink rot disease development. American Journal of PotatoResearch 86:102-11).

Several researchers have reported that mixtures of other microbialstrains can enhance and/or improve the consistency of biological control(Pierson and Weller 1994. Use of mixtures of fluorescent pseudomonads tosuppress take-all and improve the growth of wheat. Phytopathology84:940-947; Duffy and Weller. 1995. Use of Gaeumannomyces graminis var.graminis alone and in combination with fluorescent Pseudomonas spp. tosuppress take-all of wheat. Plant Disease 79:907-911; Duffy et al. 1996.Combination of Trichoderma koningii with fluorescent pseudomonads forcontrol of take-all on wheat. Phytopathology 86:188-194; Janisiewicz.1996. Ecological diversity, niche overlap, and coexistence ofantagonists used in developing mixtures for biocontrol of post-harvestdiseases of apples. Phytopathology 86:473-479; Leeman et al. 1996.Suppression of Fusarium wilt of radish by co-inoculation of fluorescentPseudomonas spp. and root-colonizing fungi. European Journal of PlantPathology 102:21-31; de Boer et al. 1999. Combining fluorescentPseudomonas spp. strains to enhance suppression of Fusarium wilt ofradish. European Journal of Plant Pathology 105:201-210; Guetsky et al.2001. Combining biocontrol agents to reduce the variability ofbiological control. Phytopathology 91:621-27; Krauss and Soberanis.2001. Biocontrol of cocoa pod diseases with mycoparasite mixtures,Biological Control 22: 149-158; Hwang and Benson. 2002. Biocontrol ofRhizoctonia stem and root rot of poinsettia with Burkholderia cepaciaand binucleate Rhizoctonia. Plant Disease 86:47-53; Cruz et. al. 2006.Exploiting the genetic diversity of Beauveria bassiana for improving thebiological control of the coffee berry borer through the use of strainmixtures, Applied Microbiology and Biotechnology 71: 916-92. Preliminaryresearch has shown that formulations containing multiple strains of theabove-mentioned dry rot antagonists performed more consistently thanindividual strains did when subjected to thirty-two storage environmentsvarying in potato cultivar, harvest year, potato washing procedure(microflora exposure), temperature, and storage time (Slininger et al.2001. Combinations of dry rot antagonistic bacteria enhance biologicalcontrol consistency in stored potatoes. Phytopathology 91:S83) However,despite these advances, the need remains for improved biological controlagents for inhibiting fungal diseases of potatoes.

SUMMARY OF THE INVENTION

We have now discovered novel compositions of bacteria which areeffective for inhibiting (reducing the incidence or severity of) fungaldiseases of potatoes, and particularly fungal diseases of potatoes understorage conditions. The bacteria which comprise the compositions of thisinvention, specifically two or more of Pseudomonas fluorescens (NRRLB-21.13), Pseudomonas fluorescens biovar (NRRL B-21053), Pseudomonasfluorescens (NRRL B-21102) and Enterobacter cloacae (NRRL B-21050), havebeen previously described for the control of fungi-induced diseases ofpotatoes. However, we have unexpectedly discovered that when two or moreof these bacteria are cultured together in the same culture medium, theresultant composition or co-culture is significantly more effective forinhibiting fungi-induced diseases of potatoes than blends of the samebacteria which have been cultured separately. In addition to theirefficacy as biological control agents against fungi-induced diseases,the compositions also exhibit significantly greater control of sproutingin stored potatoes. The method of producing the bacterial antagonisticcompositions comprises:

-   -   a) inoculating a culture medium with at least two bacteria        selected from the group of Pseudomonas fluorescens (NRRL        B-21133), Pseudomonas fluorescens (NRRL B-21053), Pseudomonas        fluorescens (NRRL B-21102) and Enterobacter cloacae (NRRL        B-21050), and incubating the inoculated medium containing said        bacteria under conditions effective for their growth, and for a        period of time effective to produce a co-culture thereof; and    -   (b) recovering the co-culture, a composition of bacteria        effective for inhibiting fungal diseases of potatoes.

In accordance with this discovery, it is an object of this invention toprovide novel compositions of bacteria which are superior antagonists offungi responsible for fungi-induced potato diseases which occur in thefield or in postharvest storage.

Another object of this invention is to provide novel compositions ofbacteria which are superior antagonists of fungi responsible forFusarium dry rot and other potato diseases which occur duringpostharvest storage.

Yet another object of this invention is to provide novel compositions ofbacteria which exhibit significantly increased efficacy for inhibitingsprouting of potatoes during storage.

A further object of this invention is to provide novel method forproducing compositions of bacteria which exhibit significantly increasedefficacy for inhibiting fungi induced diseases of potatoes andinhibiting sprouting of stored potatoes.

These and other objects of the invention will become readily apparentfrom the ensuing description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows Growth of strains P. fluorescens strains S11:P:12,P22:Y:05, and S22:T:04 in duplicate co-culture fermentor runs asdescribed in Example 1.

DEPOSIT OF BIOLOGICAL MATERIAL

Four bacterial antagonists described herein have been deposited underthe terms of the Budapest Treaty in the Agricultural Research ServiceCulture Collection (NRRL), 1815 N. University St., Peoria, Ill. 61604,USA. These four bacteria were all originally described and depositedunder the Budapest Treaty as set forth in Slininger et al., U.S. Pat.No. 5,552,315, the contents of which are incorporated by referenceherein. As described therein, Pseudomonas fluorescens biovar 5 isolateP22:Y:05 and Enterobacter cloacae S11:T:7 (which is also designatedisolate S11:3:T:06) were deposited on Feb. 22, 1993 and were assigneddeposit accession numbers NRRL B-21053 and NRRL B-21050, respectively.Pseudomonas fluorescens biovar 1 S22:T:04 was deposited on May 26, 1993,and was assigned deposit accession number (NRRL B-21102). Pseudomonasfluorescens biovar 5 isolate S11:P:12 was deposited on Aug. 30, 1993,and was assigned deposit accession number NRRL B-21133. The taxonomiccharacteristics for these deposited bacteria are described in the abovementioned U.S. Pat. No. 5,552,315.

DETAILED DESCRIPTION OF THE INVENTION

For purposes of this invention it is understood that the use of termFusarium is intended to include both the sexual (teleomorphic) stage ofthis organism and also the asexual (anamorphic) stage, also referred toas the perfect and imperfect fungal stages, respectively. For example,the anamorphic stage of Gibberella pulicaris (Fries) Sacc. is known asFusarium sambucinum (Fuckel). Fusarium-induced potato dry rot is adisease caused when a potato wound becomes inoculated with conidiaproduced by the imperfect form of this fungus.

The expression “superior antagonist” used herein in reference to amicroorganism is intended to mean that the subject strain exhibits adegree of inhibition of fungal-induced potato disease (i.e.proliferation of an agent responsible for the disease) exceeding, at astatistically significant level, that of an untreated control.

The bacterial antagonist compositions of this invention are preparedfrom Pseudomonas fluorescens (NRRL B-21133), Pseudomonas fluorescensbiovar (NRRL B-21053), Pseudomonas fluorescens (NRRL B-21102) andEnterobacter cloacae (NRRL B-21050), all of which were previouslydescribed in Slininger et al., U.S. Pat. No. 5,552,315. Although each ofthese isolates are effective for inhibiting fungi-induced diseases ofpotatoes, we have discovered that when to or more of these bacteria arecultured together in the same culture medium, the resultant compositionor co-culture exhibits significant efficacy or inhibiting fungi-induceddiseases of potatoes (i.e., they are superior antagonists). Moreover,the resultant co-cultures are significantly more effective forinhibiting fungi-induced diseases of potatoes than blends or mixtures ofthe same bacteria which are cultured separately and then subsequentlycombined or mixed. Further still, these compositions also exhibitsignificantly greater efficacy for inhibiting the sprouting of storedpotatoes than blends or mixtures of the same bacteria which are culturedseparately.

The bacterial antagonist compositions are produced by inoculating aculture medium with at least two of the above-mentioned bacteria of thegroup of Pseudomonas fluorescens (NRRL B-21133), Pseudomonas fluorescens(NRRL B-21053), Pseudomonas fluorescens (NRRL B-21102) and Enterobactercloacae (NRRL B-21050), and incubating this inoculated medium containingthe two or more bacteria under conditions effective for their growth,thereby producing a co-culture of the inoculated bacteria.

Propagation of the bacterial antagonists to prepare the composition orco-culture may be effected by culture under any conventional conditionsand in media which promote their growth. Moreover, the differentbacteria may be inoculated into the culture medium at the same ordifferent times. However, we have discovered that the bacteria must becultured together, that is, grown in the same culture medium in the sameculture vessel or fermentor, for a sufficient period of time effectiveto increase the total concentration (i.e., population of cells) of theinoculated bacteria by at least approximately one order of magnitude(i.e., the total concentration of the bacteria must increase by at leasta factor of approximately ten, measured from the total concentration ofthe bacteria in the culture medium at the time of their combination).The precise time period will of course vary with the culture conditionsand may be readily determined by the skilled practitioner usingconventional techniques to measure total cell numbers. Such techniquesmay include, for example microscopic counting, electronic particlecounting (e.g., using a Coulter counter, serial dilution cultures, andoptical cell density measurements of the culture medium. However,without being limited thereto, the minimum time of co-cultivation of thebacterial antagonists together, will typically be at least about 16hours, which time should be sufficient to allow growth of all bacterialpopulations to increase by at least one order of magnitude. The amountof bacteria used in the inoculation is not critical, although theskilled practitioner will recognize that very small inoculums mayrequire longer incubation times to produce sufficiently large quantitiesof co-culture for commercial applications. The amounts of the bacterialinoculums are typically approximately the same, however, greater amountsare preferably used for a slower growing bacterium in comparison to afast growing bacterium.

A variety of known culture media are suitable for use herein for theculture and production of the bacterial antagonists of the co-culture.As a practical matter, and without being limited thereto, the bacteriaantagonists are typically grown in aerobic liquid cultures on mediawhich contain sources of carbon, nitrogen, and inorganic saltsassimilable by the microorganism and supportive of efficient cellgrowth. Preferred carbon sources are hexoses such as glucose, but otherassimilable sources include glycerol, amino acids, xylose, etc. Manyinorganic and proteinaceous materials may be used as nitrogen sources inthe growth process. Preferred nitrogen sources are amino acids an, urea,but others include gaseous ammonia, inorganic salts of nitrate andammonium, vitamins, purines, pyrimidines, yeast extract, beef extract,proteose peptone, soybean meal, hydrolysates of casein, distiller'ssolubles, and the like. Among the inorganic minerals that can beincorporated into the nutrient medium are the customary salts capable ofyielding calcium, zinc, iron, manganese, magnesium, copper, cobalt,potassium, sodium, molybdate, phosphate, sulfate, chloride, borate, andlike ions. Similarly, suitable pH and temperature conditions are alsovariable. Cell growth of the disclosed bacteria can be achieved attemperatures between 1° and 40° C., with the preferred temperature beingin the range of 15° and 30° C. The pH of the nutrient medium can varybetween 4 and 9, but the preferred operating range is 6-8. The bacteriashould be cultivated under aerobic conditions, preferably withagitation. The total time for the culture will be dependent oncultivation conditions, particularly the culture medium, temperature,and aeration. For the purpose of illustration and without being limitedthereto, the co-culture is typically harvested 72 hr after inoculationwhen grown at 25° C., but may be as early as 20 to 24 hr, especiallywhen grown under conditions leading to more rapid growth, such as highertemperatures (26-30° C.) or on certain media with ingredients that aremore rapidly metabolized.

Following cultivation, the resultant co-culture of the bacterialantagonists is recovered for subsequent use. Although it is envisionedthat crude preparations of the co-cultured bacteria in culture media maybe used directly, in a preferred embodiment the bacteria are harvestedand formulated as described herein below.

The bacterial antagonist compositions of this invention are effectivefor controlling, that is reducing the incidence or severity of fungaldiseases of potatoes, in comparison to untreated controls. In apreferred embodiment, the compositions are effective for inhibitingfungal diseases of potatoes selected from the group consisting ofFusarium dry rot (caused by Gibberella pulicaris), pink rot (caused byPhytophthora erythroseptica, and late blight (caused by Phytophthorainfestans). In addition, the compositions are also effective for controlof (inhibiting) sprouting in stored potatoes, in comparison to untreatedcontrols.

Following recovery, the bacterial co-culture may be optionally examinedor screened to confirm efficacy in reducing the incidence and/orseverity of disease in potatoes caused by the fungal agent of interest,particularly those described herein. A variety of bioassay techniquesare suitable for use herein, such as described in the examplehereinbelow, as well as in Slininger, U.S. Pat. No. 5,552,315.Alternatively or in addition, the bacterial co-culture may be optionallyexamined or screened to confirm efficacy in reducing the incidence ofsprouting of potatoes during storage. Techniques suitable for use in theevaluation of sprout inhibition activity are described, for example, bySlininger et al., U.S. Pat. No. 6,107,247, the contents of which areincorporated by reference herein.

The bacterial compositions of the invention can be applied by anyconventional method to the surfaces of potato tuber materials, toinclude without limitation whole potato tubers, potato tuber parts, orseed tubers. For example, they can be applied as an aqueous spray ordip, as a wettable powder, or as a dust. Formulations designed for thesemodes of application will usually include a suitable liquid or solidcarrier together with other adjuvants, such as wetting agents, stickingagents and the like. Starch, polysaccharides, sodium alginate,cellulose, etc are often used in such formulations as carriers andsticking agents, and are suitable for use herein as well.

Where the desired effect is control of fungal diseases of potatoes, theexpressions “an effective amount” and “a suppressive amount” are usedherein in reference to that quantity of antagonist composition which isnecessary to obtain a statistically significant reduction in the levelof disease (measured as a decrease in the severity or the rate ofincidence) relative to that occurring in an untreated control undersuitable conditions of treatment as described herein. Without beinglimited thereto, the actual rate of application of a liquid formulationwill typically vary from a minimum of about 1×10³ to about 1×10¹⁰ totalviable cells/ml, and preferably from about 1×10⁵ to about 1×10⁹ totalviable cells/m, assuming a mode of application which would achievesubstantially uniform contact of at least about 90% of the potatosurface. If the composition is applied as a solid formulation, the rateof application should be controlled to result in a comparable number ofviable cells per unit area of potato surface as obtained by theaforementioned rates of liquid treatment.

Conversely, where the desired effect is inhibition of potato sprouting,the expressions “an effective amount” and “a suppressive amount” areused herein in reference to that quantity of the bacterial compositionthat is necessary to obtain a reduction in the amount of sprouting ofthe harvested potatoes during storage (and optionally diseaseproliferation) relative to that occurring in an untreated control undersuitable conditions of treatment and storage as described herein. Therate of application of harvested co-culture will typically be in therange of 0.05 to 2 ml of harvested culture volume per 8 oz. potato. On acell concentration basis, the rate of application should be at leastabout 1×10⁸ total viable cells/ml and preferably at least 1×10⁹ totalviable cells/ml. Even better results are obtained at concentrationsexceeding 1×10¹⁰ total viable cells/ml. The optimum dosage will dependon a number of factors, such as the size of the potato, the specificbacteria in the co-culture, and the associated cultivation conditions ofthe bacterial compositions. The skilled practitioner will be able todetermine the dosage of a given co-culture broth required for optimumexpression of sprout and dry rot biocontrol activities.

It is envisioned that the temperatures at which the bacterialcompositions are effective would range from about 5° C. to about 30° C.The preferred temperature range is 10 to 25° C., and the optimal rangeis considered to be 12° C. to 20° C. Therefore, the bacterialcompositions can theoretically be applied at any time during theharvest, grading, or shipping process, or during the early stages ofstorage. Of course, potato tubers are susceptible to infection any timea wound occurs and the fungal disease agent is present. Therefore, thelonger the delay between the tuber wounding and the treatment with thebacterial composition, the greater the chance the pathogen willsuccessfully infect the tuber. Though we have previously demonstratedthat delays of 4 h between wounding and treatment do not significantlyaffect antagonist performance, it is anticipated that longer delays maydecrease the effectiveness of the treatment depending on methods of cellformulation and application.

The following example is intended only to further illustrate theinvention and is not intended to limit the scope of the invention whichis defined by the claims.

Example 1 Materials and Methods Bacterial Antagonists

Suppressive strains Pseudomonas fluorescens biovar 5 (S11:P:12 NRRLB-21133 and P22:Y:05 NRRL B-21053), Pseudomonas fluorescens biovar 1(S22:T:04 NRRL R—21102) and Enterobacter cloacae (S11:T:07 NRRL B-21050)isolated by Schisler and Slininger (1994, ibid) were stored lyophilizedin the ARS Patent Culture Collection (NCAUR, USDA, Peoria, Ill.). Stockcultures of bacteria in 10% glycerol were stored at −80° C. Glycerolstocks were streaked to ⅕ strength trypticase soy broth agar plates (⅕TSA; Difco Laboratories, Detroit, Mich.) which were incubated 2-3 daysat 25° C. ad refrigerated up to one week as a source of precultureinoculum.

Cultivation Medium

SDCL medium was prepared with 2 g/L each K₂HPO₄ and KH₂PO₄; mineralsincluding 0.1 g/L MgSO₄(7H₂O), 10 mg/L NaCl, 10 mg/L FeSO₄(7H₂O), 4.4mg/L ZnSO₄(7H₂O), 11 mg/L CaCl₂(2H₂O), 10 mg/L MnCl₂(4H₂O), 2 mg/L,(NH₄)₆Mo₇O₂₄(4H₂O), 2.4 mg/L H₃BO₃, 50 mg/L EDTA; 0.01 g/L each ofgrowth factors adenine, cytosine, guanine, uracil, thymine; 0.5 mg/Leach of vitamins thiamine, riboflavin, calcium pantothenate, niacin,pyridoxamine, thioctic acid; 0.5 mg/L each of vitamins folic acid,biotin, B₁₂; 15 g/L Difco vitamin-free casamino acids, 0.15 g/Ltryptophan, 0.6 g/L cysteine, and 15 g/L glucose. Macro minerals, aminoacids, glucose, and acidified purines and pyrimidines were autoclavedseparately. Vitamins and trace minerals<0.1 g/L were filter sterilized.After combining sterilized ingredient groups, pH was adjusted to 7 withNaOH.

Shake-Flask Cultivations of Bacterial Inocula for Test Cultures

Fifty-mL precultures were the source of inocula for fermentors and shakeflask test cultures. Pre-cultures contained SDCL medium in 125-mLflasks, and were shaken at 250 rpm (2.5 cm eccentricity) and 25° C. in aNew Brunswick Psychroterm incubator. Cultures harvested after 24 hincubation were used to supply bacteria for baffled flask or fermentortest culture inoculations. Typical cell accumulations reached˜0.5-1×10¹⁰ per mL.

Baffled Shake-Flask Cultivations of Bacteria

Test cultures of 75-mL volume were incubated in 500-mL baffled flasksshaken at 25° C. and 250 rpm. The culture medium was the SDCL mediumabove enriched to contain 40 g/L glucose, 60 g/L casamino acids, 0.6 g/Ltryptophan, and 2.4 g/L cysteine. Bacteria were inoculated to an initialabsorbance at 620 nm of 0.1 (1×10⁸ viable cells/mL) and harvested after72 h of incubation. In flask co-cultures, the initial bacterialconcentrations were set such that each population initially contributedequally to the overall culture absorbance, except that the E. cloacaeS11:T:07 population in co-cultures was always set at 0.0001 since thisstrain could exclude the other three P. fluorescens strains ifinoculated to higher levels.

Fermentor Cultivations of Bacteria

Bacteria were cultivated in 2-L B. Braun Biostat B or ED fermentorscharged with 1.6 L of the SDCL medium above enriched to contain 40 g/Lglucose, 0 g/L casamino acids, 0.6 g/L tryptophan, and 2.4 g/L cysteine.Fermentors were controlled at 25° C., pH 7 (with 6N NaOH or 3 N HCladditives), 1 L/min aeration, and variable stirring 300-1500 rpm tomaintain dissolved oxygen at 30% of saturation. To control foaming, a20% solution of Cognis FBA 3107 was dosed as needed. Bacteria wereinoculated to an absorbance at 620 nm of 0.1 (1×10⁸ viable cells/mL) andharvested after growth 72 h; at which time, viable cell accumulationswere typically ˜2-3×10¹⁰ per mL, giving an absorbance of ˜20. Fermentorco-cultures of Pseudomonas fluorescens strains were inoculated to havethe following initial absorbances, unless otherwise specified: 0.05 foreach of S22:T:04 and S11:P:12 and 0.01 for P22:Y:05.

Enumeration of Mixed Bacteria Populations Using Selective Plating Media

Total bacterial biomass (b) was assessed using absorbance at A₆₂₀ whereb=kA and k=0.408 g/L per absorbance unit assuming a 1 cm path length(Slininger and Jackson. 1992. Nutritional factor regulating growth andaccumulation of phenazine 1-carboxylic acid by Pseudomonas fluorescens2-79. Applied Microbiology and Biotechnology 37:388-392). The viablecell concentrations of each population in mixed cultures or blends wereassessed by dilution plating cultures in duplicate to each of threeselective agar media: King's Medium (KMB), ⅕ TSA plus Tetrazolium(TSA-T), and Minimal Medium with Histidine and Tetrazolium (MMHT). Totalcounts of all strains as well as S11:P:12 were obtained from TSA-Tspread plates after 24-48 h incubation at 25° C., Strain S11:P:12 formeda distinctively large diameter diffuse shiny colony becoming creamy witha pink concentric rings (2-5 mm). Other strains formed smaller denseround colonies (1-2 mm) with red centers and white borders. A selectivecount of P22:Y:05 as deep red colonies, white edges (1 mm) was obtainedfrom MMHT spread plates after incubation 48-72 hours, while colonies ofthe other strains used in the study remained relatively tiny and white.The plating tool for P22:Y:05 was developed using Biolog GN plates toscreen carbon sources used by the four bacteria, allowing discovery ofhistidine as a selective carbon source. A specific count of Enterobactercloacae S11:T:07 was determined by picking 30 random non-S11:P:12colonies from each countable TSA-T plate to KMB plates where thepercentage of non-fluorescing colonies could be evaluated.Non-fluorescent colonies on KMB were E. cloacae, whereas fluorescentcolonies were either P. fluorescens P22:Y:05 or S22:T:04.

Plating medium ingredients in the ⅕ TSA-T were: 6 g/L Difco BactoTryptic Soy Broth, 15 g/L Difco Bacto Agar, and 0.05 g/L2,3,5-triphenyl-tetrazolium chloride (tetrazolium red, T-8877, Sigma).The medium was prepared by mixing the TSB and agar in distilled water,autoclaving, then mixing in 10 mL of tetrazolium filter-sterilizedconcentrate to the ˜60° C. agar mix. Solidified plates were refrigeratedto preserve the tetrazolium red.

Ingredients in the MMHT were the following: 2 g/L K₂HPO₄, 2 g/L KH₂PO₄,0.01 g/L FeSO₄(7H₂O) 0.1 g/L MgSO4(7H₂O), 0.01 g/L NaCl, 0.0044 g/LZnSO₄ (7H₂O), 0.011 g/L CaCl₂ (2H₂O), 0.01 g/L MnCl₂(4H₂O), 0.002 g/L(NH₄)₆MO₇O₂₄(4H₂O), 0.024 g/L H₃BO₃, 0.05 g/L EDTA, 1.26 g/L urea, 5 g/Lhisitidine, 15 g/L BD Difco agar, and 0.05 g/L2,3,5-triphenyl-tetrazolium chloride. The MMHT was prepared by mixingwarm, double-strength autoclaved agar with the double-strengthfilter-sterilized nutrient solution and tetrazolium concentrate in asterile bottle with stir bar. The MMHT nutrient solution preparation wasdone similarly to the SDCL fermentation medium described above andadjusted to pH 7 before mixing with the agar solution. The medium waspoured to plates immediately upon mixing, and the solidified plates, asfor TSA-T, were stored refrigerated.

Ingredients in King's Medium B were: 10 q/L BD Difco Bacto ProteosePeptone#3, 10 g/L glycerol, 1.5 g/L K₂HPO₄, 1.5 g/L MgSO₄(7H₂O), and 15g/L BD Difco Bacto Agar. KMB ingredients were mixed, pH adjusted to 7.2,autoclaved and then molten agar was poured to plates.

Peoria Wounded Potato Bioassay of Fusarium Dry Rot or Pink RotSuppression

The wounded potato bioassay of treatment efficacy against Gibberellapulicaris (Fr.:Fr.) Sacc. (anamorph: Fusarium sambucinum Fuckel) strainR-6380 was originally described in Schisler and Slininger (1994. ibid).In the present study, the bacteria treatments were diluted by mixing 0.5mL culture with 17.5 mL of chilled buffer, and then 1:1 (v/v) with G.pulicaris R-6380 at either 1 or 3×10⁶ conidia/mL (by hemacytometercount), pending virulence in prior assays. Potato wounds made with a 2mm diameter×2 mm length steel pin were thus co-inoculated with treatmentand pathogen by pipetting 5 μL of the 1:1 (v/v) treatment:pathogenmixture to each wound. Each bacteria treatment was repeated on six sizeB Russet Burbank seed potatoes (Wisconsin Seed Potato CertificationProgram, University of Wisconsin Madison, Antigo, Wis.) that had beenwashed and dried a day ahead at room temperature, following priorstorage in a cold room ˜4° C. Each potato had four wounds equally spacedaround the middle-three wounds receiving bacteria and pathogen and onecontrol wound receiving only pathogen mixed with buffer controls. Eachpotato was placed in a plastic weigh boat on a dry 2.54 cm-cut square ofWyp-all L40 all purpose wiper paper towel (Kimberly-Clark Worldwide,Inc.). Boats were held in trays that were supplied two dry Wyp-alls overthe top of potatoes and two Wyp-alls wet with 40 mL of water each andplaced on either side of the tray, plastic bagged, and stored 21 daysat >90% relative humidity and 15° C. After storage each potato wasquartered, slicing through the center of each of the four wounds. Theextent of disease in each wound was rated by adding the greatest depthand width measurements (mm) of discolored necrotic tissue extendingbelow and to the sides of the wound. Relative disease (%) was calculatedas 100×(wound disease rating/average disease rating of wounds receivingpathogen only).

Similarly, pink rot suppression was assayed on wounded potatoes.Zoospores of the causative pathogen Phytophthora erythroseptica strain02-05 were produced per Schisler et al. (2009. Bacterial antagonists,zoospore inoculums retention time and potato cultivar influence pink rotdisease development. American Journal of Potato Research 86:102-111) andsuspended at 3×10⁴ zoospores/mL buffer before mixing 1:1 with biocontrola gent treatments (or buffer control). Then 5 μL of eachtreatment-pathogen mixture was applied to 10 replicate wounds, eachwound on a different potato. Each potato had two wounds and was used totest two treatments. Tubers were stored as for the dry rot assay, andpink rot development was rated after one week, using the same lesionwidth plus depth method as described above for dry rot.

Kimberly Small Pilot Evaluations of Biocontrol Efficacy-Late Blight,Pink Rot, Sprouting

Late Blight

Phytophthora infestans JMUIK-2002 (US-8, mating type A2) was obtainedfrom Dr. Jeff Miller (University of Idaho, Aberdeen, Id.) for smallpilot testing of disease suppression under commercial storage conditionsat the University of Idaho Kimberly Research and Extension Center,Kimberly, Id. Kimberly rye agar plates were grown in darkness for 2weeks at 18° C., where rye agar was prepared as follows: 60 g rye seedwere soaked over night; seeds were then ground in blender 3 min andstrained out using four layers cheesecloth; deionized water was pouredthrough pulp until 1 L total volume suspension was collected; 10 gglucose and 15 g agar were added just prior to autoclaving. Plates wereharvested by adding 10 mL of ˜4° C. water per plate and scraping with aglass hockey stick to recover about 9 mL containing 2×10⁵ sporangia/mL.For tuber inoculation, 2.5 L of 4×10⁴ sporangia/mL were chilled 1.5 hand then warmed to room temperature ˜15-18° C. for 45 minutes toliberate zoospores.

The suspension of P. infestans was sprayed onto all of the tubers forthe trial at a rate of 1.6 mL per 8 oz. tuber (0.8 mL/tuber first to oneside plus 0.8 mL/tuber to the other side). An air-assist syringe sprayerwas used with a Delevan brass nozzle (#4, 80° LF.). The sprayer andnozzle were sterilized between treatments by rinsing once with 70%ethanol and then three times with distilled water. Potatoes were sprayedin groups of about one hundred spread out on plastic sheeting whichcould be drawn up to cover the potatoes to retain moisture until thebacterial treatments or water controls were applied. Pathogen-inoculatedpotatoes were collected into three replicate groups, and allowed to setno more than 45 minutes prior to treatment with biocontrol agents.Bacterial cultures were harvested from fermentors and storedrefrigerated or in chilled shipping coolers 2-5 days before the trial.On the day of the trial, cultures were diluted in half with colddistilled water and returned to the refrigerator until application. Eachbacterial treatment or water control treatment was then similarlysprayed at a rate of 0.8 mL per tuber to 30 potatoes from each of thethree pathogen-treated replicate groups.

Each treatment replicate of 30 tubers was placed in a unventilatedplastic box with lid and positioned randomly on shelves in the storagebay which was maintained at 7.2° C. (45° F.) and 95% relative humidity.After four weeks storage, potatoes were peeled and rated based on thepercentage of surface area showing the discoloration typical of lateblight infection.

Pink Rot

Phytophthora erythroseptica strain 01-21, the causative pathogen of pinkrot disease, was produced by Mr. Shane Clayson and Dr. Jeff Miller(University of Idaho, Aberdeen, Id.) (Schisler et al. 2009 ibid). Torelease zoospores, plates were chilled on ice one hour and then allowedto warm to room temperature for 2 hours. To prepare pathogen inoculum tobe used in each trial, around 50 plates of P. erythroseptica (Pe) wereharvested at a rate of about 5×10⁵ zoospores/10 mL distilled water perplate.

Russet Burbank tubers were bruised by tumbling about 75 tubers at a timefor two minutes in a padded plastic cement mixer. Enough tubers werebruised to accommodate 4 reps of 15 tubers per each treatment. Pesuspension (10⁴-10⁵ zoospores/mL pending virulence) was sprayed ontotubers at a rate of 1.6 mls per 8 oz Russet Burbank or Russet Norkotatubers. Seventy-five tubers at a time were placed on a plasticsheet-covered spray table, and 60 mls were applied to the first side ofthe potatoes. Tubers were flipped and 60 more mls were applied to theother side, for a total of 120 mL per 75 tubers, or 1.6 mL per tuber.Tubers were then taken off the table using the plastic sheet, set on thecement floor (˜60-65° F.) and the edges pulled up to completely coverthe tubers and retain moisture. This process was repeated to preparefour pools of bruised, pathogen treated tubers, such that one 15-tuberreplicate of each treatment was drawn from each of the four pools.Before experimental bacterial treatments were applied, tubers were leftfor approximately 15 minutes after the final rep of each cultivar wastreated with pathogen. This process was then repeated in its entiretyfor the Russet Norkota tubers after bacterial inoculation of the RussetBurbank cultivar was complete.

Bacterial treatments and water controls were applied at a rate of 0.8 mLper tuber. As for the late bight evaluation, ready-to-spray bacterialtreatments were prepared and set aside in the refrigerator for platingat the end of the day. To form each treatment rep, 15 tubers from eachof the four plastic sheets on floor were placed onto spray table,marking the replicate origin. To one side of the 60 potatoes, 24 mlswere applied to the 60 tubers, and then another 24 mls were appliedafter the tubers were flipped. Tubers were then placed into theirrespective rep bags at 15 tubers per bag and then all 4 rep bags placedin the appropriate box. Boxes were then moved immediately into storagebay and a lid placed on the box. No ventilation was connected to theboxes. The process was then repeated with Russet Norkota being treatedwith pathogen, 15 minutes wait, then bacterial treatments.

Treatment boxes were stored at 17° C. for 3 weeks to allow diseasedevelopment. At the end of the storage period, potatoes were rated bypeeling and evaluating the percentage of surface coverage by lesionstypical of pink rot.

Sprouting

Biological control treatments and water only control were sprayed at.0.8 mL per 8 oz Russet Burbank potato. For each treatment, threereplicate groups of 70 potatoes were sprayed. Each replicate was spreadover a plastic sheet-covered table and sprayed with half the volume onone side, flipped, and then the other half of the volume was sprayed tothe other side. Each replicate was transferred to a plastic box labeledwith treatment and replicate numbers. The plastic sheet was toweled offto minimize excess collection of treatment on the plastic betweenspraying each replicate and disposed after each treatment was finished.Each box of potatoes was placed randomly in the storage, which was heldat 45° F. and 95% relative humidity with 1 cfm/cwt air circulation.Ready-to-spray treatment samples were refrigerated until plating afterfinishing all treatment applications. Treatments were monitored bydrawing 20 tubers per replicate box and measuring longest sprout pertuber, ten-potato total sprout length and ten-potato total sprout weightper replicate sampled. Potatoes were stored in November and finallymonitored in April.

Statistical Analysis

Analysis of variance (ANOVA) was performed using Sigmastat 2.03 (SPSS,Inc.) to determine significant min effects and interactions of thevariables tested. Pair-wise comparisons were made using Student NewmanKeuls (SNK) or Least Significant Difference (LSD) analysis. Thesignificance criterion applied was generally P≦0.05, or otherwise notedin the 0.1 to 0.001 range.

Results and Discussion

In both laboratory and small pilot testing of bioefficacy, co-culturedmicrobial biocontrol strains consistently outperformed pure stains andblends of strains produced individually in pure cultures.

Co-Culture Versus Blend Efficacy for all Combinations of Four StrainsGrown in Baffled Flasks

In this experiment design all 4-, 3-, and 2-strain combinations as wellas pure strains S11:P:12, S22:T:04, P22 Y:5, and S11:T:07 wereinoculated to baffled flask cultures. The co-culture and pure culturepopulations harvested after 72 h had the strain compositions andabsorbances listed in Table 7. Pure stain cultures were blended in equalvolume to Form the “blend” compositions listed. These treatments weretested in two dry rot wounded potato bioassay experiments, one at0.5×10⁶ F. sambucinum conidia/mL and another experiment at 1.5×10⁶conidia/mL per 5 μL wound. The impact of strain composition versuscombination method on Fusarium dry rot disease development (percentrelative disease) was tested and analyzed using two-way analysis ofvariance.

Selective plating media not requiring antibiotic markers were designedand used since preliminary experiments indicated that marked strains hadthe complications of growth rates and potentially metabolisms beingdifferent from parent strains and phenotype drift occurring in theabsence of antibiotic pressure. A nutrient-based selective platingscheme was chosen to monitor the individual strain populations inco-cultures, allowing results directly representing the naturalbiocontrol strains and their kinetic characteristics.

Disease ratings observed in wounded potatoes bioassays indicated thatall biocontrol agent treatments, including co-cultures, blends, and purecultures significantly reduced dry rot disease development relative toboth low and high level pathogen challenges (P<0.001) Since thetreatment×pathogen level interaction was not significant, the twopathogen level data sets were combined for further analysis. Subsequenttwo-way analysis of variance of relative disease with strain compositionof treatments by the method of combining strains (blend versusco-culture) showed that significantly better Fusarium dry rotsuppression was obtained by co-cultures than by similar strain mixturescreated by blending pure strain cultures (P<0.001) (Table 6). The meanrelative disease rating (±the standard error) for blended straincombinations was 41.3±2.4%, while only 30.3±2.4% for co-cultures,indicating that co-cultures reduced disease to a greater extent (69.7%)than had the blended strain combinations (58.7%). Using a P<0.05significance criterion, the variation of relative disease among thevarious strain compositions was not significant (P=0.082) nor was thecomposition×combination method interaction (P<0.0.76).

An analysis was also done to determine whether biocontrol strainpopulations in co-cultures and blends were performing additively orsynergistically co-culture refers to the technique of cultivatingmultiple strains together in one fermentation vessel starting from a lowinitial cell population density that multiplies and advances throughgrowth phase to stationary phase. This process contrasts with blendinglarge populations of strains together after they are grown separately aspure strain cultures from low initial cell concentration through growthphase to stationary phase. When strain populations are grown togetherprior to application, they may have the opportunity to stimulate oneanother in such a way as to provide a final mixed population thatsuppresses disease more efficiently than a blend of pure cultures. Onthe other hand, co-cultured strains may not be compatible with oneanother, such that low population concentrations result or the endproduct does not suppress disease as well as observed for pure strainsor a blend of pure strains. To test positive versus negative impact ofstrain combinations, the expected relative disease rating was calculatedfor comparison with the actual disease rating measured for eachtreatment comprised of multiple strains:RDR_(calc)=(RDR₁×N₁+RDR₂×N₂+RDR₃×N₃+RDR₄×N₄)/(N₁+N₂+N₃+N₄), where N₁ isthe concentration of strain population 1 (such as S11:P:12 viablecells/mL) in a multiple strain mixture and RDR₁ is the relative diseaserating observed for the pure strain population 1, and so forth forstrains 2 through 4. Thus, RDR_(calc) for a mixed population is apopulation-weighted average of the observed disease ratings forsingle-strain treatments; or in other words, it is addition of thefractional performance contributions of each component strain.Comparison of RDR_(calc) with actual disease ratings of strain blendsand co-cultures will indicate if the efficacy of strain components of amixture are additive or if the method of combining them provides asynergistic advantage or disadvantage or disadvantage relative to thedisease suppressiveness observed for pure strains applied individually.

After 72-h incubation both pure and co-cultures accumulated virtuallythe same biomass concentration as indicated by culture absorbancesaveraging 16.0±0.88 and 15.9±1, respectively Table 7). In co-culturescontaining E. cloacae strain S11:T:07, it was the dominant population in72-h cultures, but low concentrations of other strains also existed. Inco-cultures without E. cloacae, the 72-h populations were more equallydivided among the P. fluorescens strains present. Nearly allco-cultures, regardless of the population distribution among strains,exhibited lower disease levels than expected by calculation ofRDR_(calc) from the additive performance contributions of pure strains,The mean actual relative disease rating (±standard deviation) forco-cultures (30.3±6.3%) was significantly lower than both the meanco-culture RDR_(calc) and the mean actual relative disease rating forblends, 42.9±5.3% and 41.3±13.1%, respectively. In contrast, the meanactual relative disease rating for blends (41.3±13.1%) was similar tothat of pure strains (39.9±6.6%) as well as the predicted mean value ofRDR_(calc) for blends (40±2.8%) obtained by adding the efficacycontributions of pure strains. These findings provide evidence thatco-culturing strains stimulates synergistic inter-strain activities thatboost biocontrol efficacy. This synergy is apparently not developed instrains that are grown separately and mixed just prior to addition topotato wounds.

Consistency of Co-Culture, Blend and Pure Strain Biocontrol TreatmentEfficacy Across Lab Bioassays and Small Scale Commercial StorageSimulation

In the investigation of the various strain combinations grown in baffledflasks, final treatment populations were not always equally distributedamong the component strains in co-cultures where variable growthkinetics of strains occurred (Table 7). Similar populations in threestrain mix fermentations after 72-h cultivation were achievable infermentors by inoculating faster growing population P22:Y:05 to lowerinitial cell density than slower growing P. fluorescens strains S22:T:04and S11:P:12 (FIG. 1). The performance of this three-strain co-culturewas compared versus pure strains and the corresponding three-strainblend comprised of an equal volume mix of the three pure-strainpopulations grown under the same fermentor conditions. These treatmentswere compared in sixteen experiments conducted in laboratory experimentsin Peoria using wounded potato bioassays of dry rot or pink rotsuppressiveness or in Kimberly, Id. using small pilot simulations ofcommercial storage conditions during challenges of late blight, pinkrot, and sprouting. The performance of treatments was then ranked withineach experiment by calculating the dimensionless relative performanceindex of each treatment. Overall treatment performance could then beassessed and analyzed using analysis of variance and pairwise comparisontechniques to determine superior treatments.

Tables IA and IB show the average strain populations in ready-to-applytreatments for the 16 laboratory and small pilot experiments as measuredby dilution plating on selective media. These data show that co-culture,blend and pure strain treatments had similar total cell concentrations,and that blend and co-culture treatments had similar straindistributions. Table 2 summarizes the performance of the treatments innine small-scale pilot tests in Kimberly, Id. simulating commercialstorage conditions. Table 3 summarizes the performance of similartreatments applied in seven laboratory experiments conducted in Peoria,Ill. using the wounded potato bioassay. Scanning these data, it isevident that at least one and usually more biocontrol treatmentssignificantly reduced disease or sprouting relative to the control.Co-culture had a lower mean disease rating than the blend in 9 of 16experiments. The co-culture led other treatments in incidence ofsuccessful significant disease or sprout reduction relative to thecontrol: 14 of 16 attempts for co-culture, 11 of 16 attempts for blend,10 of 13 attempts for S11:P:12, 8 of 13 attempts for S22:T:04, and 9 of13 attempts for P22:Y:05.

For each bacterial and control treatment within an experiment, arelative performance index (RPI) was calculated, as listed in bracketsto the right of means in Tables 2 and 3. RPI is a dimensionless valuethat is useful in combining data sets to use in overall ranking orstatistical analysis of treatments submitted to various testingprocedures. Given disease or sprout ratings normally distributed acrossthe group of bacteria stains tested, the value of F=(X−X_(avg))/s rangesfrom −2 to +2. Here, X designates a disease or sprout rating observedper treatment, and X_(avg) and s are the average and standard deviation,respectively, of all values observed for the group of bacteriatreatments within a given experiment, such as the fall 2005 late blightbioassay at Kimberly. Since F decreases as disease or sproutsuppressiveness improves, then RPI=(2−F)×100/4, such that the value ofRPI ranges from ˜0 to 100 percentile from least to most suppressive,respectively.

A one-way analysis of variance of RPI by treatment showed significantefficacy of biocontrol agents to suppress potato maladies relative tothe control (Table 4). However, high standard deviation preventedstatistical separation of the performances of biocontrol treatmentsalthough co-culture treatments performed most consistently across allsixteen assays as indicated by exhibiting the highest overall mean RPIand the lowest relative standard deviation. A two-way analysis ofvariance in RPI with treatment and malady was performed which consideredco-culture, blend and control across all sixteen experiments conductedin Kimberly and Peoria. The result of this analysis is given in Table 5and indicates that the overall mean RPI for the co-culture treatment wassignificantly higher than that for the blend.

It is understood that the foregoing detailed description is given merelyby way of illustration and that modifications and variations may be madetherein withhout departing from the spirit and scope of the invention.

TABLE 1 Average biocontrol strain populations applied in treatments A.Peoria laboratory wounded potato bioassays Average PopulationConcentration × 10⁻⁸ (cells/mL)^(a,b) Treatment S11:P:12 S22:T:04P22:Y:05 Total Co-culture 0.63 ± 0.15 B 1.49 ± 0.84 B 0.61 ± 0.55 B 2.72± 0.85 A Blend 0.56 ± 0.34 B 0.84 ± 0.25 B 0.86 ± 0.26 B 2.26 ± 0.49 AS11:P:12 1.61 ± 0.74 A 0 0 1.61 ± 0.74 A S22:T:04 0 2.60 ± 1.55 A 0 2.60± 1.55 A P22:Y:05 0 0 2.28 ± 1.19 A 2.28 ± 1.19 A Control 0 0 0 0 B.Kimberly small pilot bioassays simulating commercial storage conditionsAverage Population Concentration × 10⁻⁹ (cells/mL)^(a,b) TreatmentS11:P:12 S22:T:04 P22:Y:05 Total Co-culture 2.22 ± 0.88 B 2.01 ± 2.8 B4.52 ± 2.24 B 8.76 ± 3.50 B Blend 1.48 ± 0.64 B  1.47 ± 1.40 B 2.39 ±0.76 B 5.35 ± 2.72 BC S11:P:12 3.65 ± 1.86 A 0 0 3.65 ± 1.86 C S22:T:040 12.3 ± 1.5 A 0 12.3 ± 1.5  A P22:Y:05 0 0 7.24 ± 5.70 A 7.24 ± 5.70 BCControl 0 0 0 0 ^(a)Within columns, values not sharing a similar letterare significantly different based on Student-Newman-Keuls pairwisecomparison method using P < 0.05 significance criterion. ^(b)Standarddeviations in mean values are indicated following the “±” symbol.

TABLE 2 Summary of treatment performance in Kimberly, Idaho small pilottests simulating commercial storage Late Blight (% SurfaceCoverage)^(e,h) Treatment Fall 2005 Winter 2006 Winter 2007 Fall 2007Winter 2008 Coculture 29.4[81] A 6.7[60] AB 6.3[84] A 12.1[68] A2.50[51] A Blend 45.3[49] B 5.8[74] A 11.2[40] AB 13.4[55] A 1.85[60] AS11:P:12 — — — — 6.9[78] A 15.4[34] A 1.63[63] A S22:T:04 — — — —11.1[41] AB 11.9[70] A 1.39[66] A P22:Y:05 — — — — 10.5[47] AB 11.7[72]A 1.52[65] A Control^(d) 59.4[20] C 9.5[16] B 14.7[10] B 18.6[2] B6.38[−5] B Significance P < 0.05 P < 0.075^(a) P < 0.05^(a) P < 0.1^(a)P < 0.05^(a) Pink Rot (% Surface Coverage)^(f,h) Treatment Winter2007^(g) Winter 2008 RB Winter 2008 RN Co-culture 50.8[54] AB 66.3[53]AB 90.8[69] A Blend 48.6[60] AB 59.3[73] A 89.3[80] A S11:P:12 40.9[81]A 75.8[26] BC 92.2[58] A S22:T:04 53.8[46] B 59.8[72] A 95.1[37] ABP22:Y:05 48.6[60] AB 60.8[69] A 92.8[54] A Control^(d) 70.6[0] C 82.5[7]C 99.8[2] B Significance P < 0.05^(b) P < 0.05^(b) P < 0.1^(c) SproutWeight (%)^(h) Treatment Fall 2005-April 2006 Co-culture 0.438[80] ABlend 0.603[51] AB Control^(d) 0.788[19] B Significance P < 0.05^(a)^(a)Within the column, values having no letters in common aresignificantly different based on Student-Newman-Keuls pairwisecomparison test. ^(b)Within the column, values having no letters incommon are significantly different based on Fisher's Protected LSD testusing arcsine transformation if needed to normalize data. ^(c)Values notsharing a similar letter are significantly different based on anunprotected LSD test. ^(d)The control treatment consists of waterinstead of biocontrol agent applied to potatoes infested with pathogen.^(e)Late blight is incited by 4 × 10⁴ sporangia/mL Phytophthorainfestans sprayed at 1.6 mL per 8 oz Russet Burbank tuber. ^(f)Pink rotis incited by 10⁴-10⁵ zoospores/mL, pending virulence, Phytophthoraerythroseptica sprayed at 1.6 mL per 8 oz Russet Burbank (RB) or RussetNorkota (RN) tubers. ^(g)Data represent combined means of Russet Burbankand Russet Norkota data sets since no significant treatment x cultivarinteractions. ^(h)Each number in brackets indicates the correspondingrelative performance index (RPI) of the value relative to other datawithin the experiment column.

TABLE 3 Summary of treatment performance in Peoria laboratory woundedpotato bioassays Dry Rot Disease Rating (mm)^(a,g) Treatment Fall2006^(e) Winter 2007^(e2) Spring 2007A^(e) Spring 2007B^(e) Fall2007^(e2) Co-culture 5.3[63] A 5.2[71] A 23.0[62] A 28.7[77] A 6.7[62] ABlend 6.2[59] A 5.5[71] A 27.7[53] A 27.2[82] A 6.2[64] A S11:P:124.5[63] A 9.3[64] A 27.8[52] A 35.0[53] AB 9.0[54] A S22:T:04 3.2[65] A19.3[46] A 16.3[75] A 35.3[52] AB 6.8[62] A P22:Y:05 7.2[57] A 17.8[49]A 23.3[61] A 44.1[19] AB 6.0[64] A Control^(d) 37.6[−6] B 46.1[−1] B56.3[−3] B 44.2[18] B 26.9[−5] B Significance P < 0.001^(b) P <0.001^(b) P < 0.001^(b) P < 0.05^(b) P < 0.001^(b) Pink Rot DiseaseRating (mm)^(a,f,g) Treatment Fall 2006 Winter 2007 Co-culture 54.5[39]AB 19.5[83] A Blend 57.9[24] B 23.4[45] ABC S11:P:12 43.3[89] A 20.0[78]ABC S22:T:04 53.2[44] AB 26.9[11] C P22:Y:05 45.7[78] AB 23.0[49] ABCControl^(d) 57.7[25] B 24.6[33] BC Significance P < 0.1^(c) P < 0.1^(c)^(a)Disease ratings represent lesion width plus depth around a wound.^(b)Values not sharing a similar letter are significantly differentbased on Student-Newman-Keuls Method pairwise comparison method.^(c)Values not sharing a similar letter are significantly differentbased on an LSD all pairwise comparisons test applied to the diseaseratings. ^(d)The control treatment consists of water + pathogen onlyapplied to potatoes infested with pathogen. ^(e)Dry rot is incited by0.5 × 10⁶ conidia Fusarium sambucinum per mL pipetted 5 μmL per wound onRusset Burbank potatoes. ^(e2)Dry rot is incited by 1.5 × 10⁶ conidiaFusarium sambucinum per mL pipetted 5 μmL per wound on Russet Burbankpotatoes. ^(f)Pink rot is incited by 1.5 × 10⁴ zoospores Phytophthoraerythroseptica pipetted 5 μmL per wound. Data represent combined meansof Russet Burbank and Russet Norkota data sets for each treatment.^(g)Each number in brackets indicates the corresponding relativeperformance index (RPI) of the value relative to other data within theexperiment column.

TABLE 4 One-way analysis of variance by treatment shows significantefficacy of biocontrol agents to suppress potato maladies relative tothe control, but high standard deviation prevents performancedifferentiation when interactions of treatment by potato malady type areignored. Co-culture treatments perform most consistently across allsixteen assays with lowest relative standard deviation. StandardRelative Deviation Standard Treatment Mean RPI^(a) In Mean RPI Deviation(%) Co-culture 65.9 A 13.1 19.8 Blend 58.7 A 15.4 26.2 S11:P:12 61.1 A18.1 29.6 S22:T:04 52.9 A 17.9 33.8 P22:Y:05 57.2 A 14.9 26.0 Control8.2 B 12.2 148.7 ^(a)Within the column, means not sharing a similarletter are significantly different based on the Student-Newman-Keulspairwise comparison method (P < 0.05 significance criterion).

TABLE 5 Two-way analysis of variance of Relative Performance Index (RPI)with treatment x malady indicated that the relative biocontrolperformance of the co-culture treatment was significantly better thanthat of the blend treatment over all sixteen experiments conducted inKimberly and Peoria. Average RPI Average RPI Potato Malady Treatment xfor Treatment Treatment Bioassay Malady^(a) Groups^(a,b) Co- Late Blight68.7 ± 6.2 68.7 ± 4.4 A culture Pink Rot 59.6 ± 6.2 Dry Rot 66.5 ± 6.2Sprouting  80.0 ± 13.9 Blend Late Blight 55.6 ± 6.2 57.2 ± 4.4 B PinkRot 56.4 ± 6.2 Dry Rot 65.7 ± 6.2 Sprouting  51.2 ± 13.9 Control LateBlight  8.5 ± 6.2 10.3 ± 4.4 C Pink Rot 18.8 ± 6.2 Dry Rot 13.5 ± 6.2Sprouting  0.5 ± 13.9 ^(a)Least square means are reported, and thestandard error in the means is given following each “±” symbol.^(b)Within the column, values having no letters in common aresignificantly different based on Student-Newman-Keuls pairwisecomparison test using the P < 0.075 significance criterion.

TABLE 6 Two-way analysis of variance of relative disease with straincomposition of treatments and method of combining strains showed thatsignificantly better Fusarium dry rot suppression was obtained by strainco-cultures than by similar strain mixtures created by blending purestrain cultures. Treatment Composition of Strains Least Square Mean ofStrain Presence (1) Absence (0) Relative Disease (%)^(a,b) PseudomonasPseudomonas Pseudomonas Enterobacter Strain Combination Methodfluorescens fluorescens fluorescens cloacae Co- S11:P:12 S22:T:04P22:Y:05 S11:T:07 culture Blend 1 1 1 1 28.2 25.4 1 1 1 0 39.8 30.8 1 01 1 39.8 50.5 1 1 0 1 30.7 34.3 0 1 1 1 22.5 45.5 1 0 1 0 25.0 44.8 1 10 0 26.7 30.0 1 0 0 1 36.8 32.2 0 1 1 0 23.5 48.0 0 0 1 1 27.1 40.6 0 10 1 33.1 71.9 Overall Least Square Means ± Standard Error^(b) 30.3 ± 2.4A 41.3 ± 2.4 B Significance P < 0.001 ^(a)All treatments were challengedwith Fusarium sambucinum conidia at 0.5 or 1.5 × 10⁶ per mL pipetted 5μL per Russet Burbank potato wound. Dry rot lesions in controls withoutBCA had disease ratings averaging 10.5 or 13 mm, respectively pendingpathogen inoculum size. Since relative disease did not varysignificantly with the interaction of treatment x pathogenconcentration, analysis was conducted on the combined low and high levelpathogen data sets. ^(b)The overall least square means of relativedisease observed for strain co-cultures versus blends were significantlydifferent (P = 0.001) based on Student-Newman-Keuls pairwise comparisonmethod as designated by different letters. Relative disease did not varysignificantly due to the strain composition of mixed strain treatmentsor the interaction of treatment strain composition x combination method.

TABLE 7 One-way analysis of variance of actual and calculated relativedisease ratings indicate that strains grown in co-cultures developsynergistic activities that support superior disease suppressivenesscompared with blends of pure strains cultivated separately. 72-h ActualCalculated Strain 72-h Culture Viable Cell Culture Relative DiseaseRelative Disease Combination Populations × 10⁻¹⁰ (cells/mL)^(e)Absorbance Rating^(a,b,c) Rating^(a,b) Method S11:P:12 S22:T:04 P22:Y:05S11:T:07 (620 nm)^(a) (%) (%) Co-culture 0.25 0.0018 0.10 2.4 15.2 28.246.2 0.40 0.56 1.1 0 16.4 39.9 35.9 0.027 0 0.046 1.1 14.7 39.9 46.50.16 0.0055 0 2.2 16.3 30.7 46.8 0 0.038 0.10 1.8 16.6 22.5 46.1 0.44 00.57 0 16.3 25.0 37.8 0.29 1.1 0 0 17.4 26.7 37.3 0.0031 0 0 0.23 17.336.9 47.0 0 0.42 0.56 0 14.4 23.5 34.2 0 0 0.017 2.5 15.5 27.1 47.0 00.012 0 2.2 15.2 33.1 47.0 Means 15.9 ± 1.0 A  30.3 ± 6.3 A, a  42.9 ±5.3 A, b Blend^(d) 0.28 0.4 0.085 0.2 25.4 40.2 0.37 0.53 0.11 0 30.838.4 0.37 0 0.11 0.27 50.5 43.4 0.37 0.53 0 0.27 34.4 40.9 0 0.53 0.110.27 45.5 38.6 0.55 0 0.17 0 44.9 41.4 0.55 0.80 0 0 30.0 39.0 0.55 0 00.40 32.2 45.3 0 0.80 0.17 0 48.0 35.2 0 0 0.17 0.40 40.6 42.9 0 0.80 00.40 71.9 39.4 Means 41.3 ± 13.1 B, a 40.4 ± 2.8 A, a Pure 1.1 16.4 44.044.0 1.6 14.7 35.6 35.6 0.34 16.3 33.1 33.1 0.8 16.6 47.1 47.1 Means16.0 ± 0.88 A 39.9 ± 6.6 AB   39.9 ± 6.6 A  ^(a)Within columns, theoverall relative disease or culture absorbance means ± standarddeviation shown in bold for co-culture, blend and pure strain treatmentsare significantly different if there are no “capital” letters in commonbased on Student-Newman Keuls pairwise comparison with significancecriterion of P < 0.05. ^(b)Within rows, the overall means for actualversus calculated relative disease are significantly different if thereare no “lower case” letters in common based on a paired t-test withsignificance criterion P < 0.05. ^(c)Since relative disease did not varysignificantly with treatment strain composition or treatment x pathogenconcentration interactions, statistical one-way analysis of variance wasconducted on the combined low and high level pathogen experiment datasets (0.5 or 1.5 × 10⁶ Fusarium conidia/mL in 5 μL inoculations toRusset Burbank potato wounds). Fusarium dry rot lesions in controlswithout biocontrol agent had disease ratings averaging 10.5 or 13 mm,for low and high level disease challenge respectively, whichcorresponded to 100% relative disease. ^(d)Viable cell concentrations ofblend treatments were calculated from the pure stain plate countsreported since equal volume mixtures of pure strain cultures wereprepared. ^(e)Strain populations listed as 0 cells/mL were not presentin the co-culture inocula, or they were not included in blends. Theconcentrations listed are prior to dilution 72 times for the woundedpotato bioassay.

1. (canceled)
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 9. A composition of bacteriaproduced by the process comprising: a) inoculating a culture medium withat least two bacteria selected from the group consisting of Pseudomonasfluorescens deposited at the Agricultural Research Service CultureCollection (NRRL) under deposit accession number NRRL B-21133,Pseudomonas fluorescens deposited at the Agricultural Research ServiceCulture Collection (NRRL) under deposit accession number NRRL B-21053,Pseudomonas fluorescens deposited at the Agricultural Research ServiceCulture Collection (NRRL) under deposit accession number NRRL B-21102and Enterobacter cloacae deposited at the Agricultural Research ServiceCulture Collection (NRRL) under deposit accession number NRRL B-21050,and incubating the inoculated medium containing said bacteria underconditions effective for growth thereof and for a period of timeeffective to produce a co-culture of said bacteria; and (b) recoveringsaid co-culture.
 10. The composition of bacteria of claim 9 wherein saidbacteria are incubated together in said culture medium in (a) underconditions and for a period of time effective to increase the totalconcentration of said bacteria by at least approximately one order ofmagnitude.
 11. The composition of bacteria of claim 9 wherein saidprocess further comprises screening said co-culture from (b) to selectfor effectiveness for inhibiting fungal diseases of potatoes.
 12. Thecomposition of bacteria of claim 9 wherein said process furthercomprises screening said co-culture from (b) to select for effectivenessfor inhibiting sprouting of potatoes.
 13. A method for suppressingfungal diseases in potatoes comprising applying to whole potato tubers,potato tuber parts, or seed tubers, the composition of bacteria of claim9 in an amount effective to reduce the level of fungi-induced disease inthe potato relative to an untreated control.
 14. The method of claim 13wherein said fungi-induced disease is selected from the group consistingof Fusarium dry rot caused by Gibberella pulicaris, pink rot caused byPhytophthora erythroseptica, and late blight caused by Phytophthorainfestans.
 15. A method for suppressing sprouting in stored potatoescomprising applying to a potato tuber the composition of bacteria ofclaim 9 in an amount effective to reduce the level of sprouting in thepotato tuber during storage relative to an untreated control.